In the Supernatural episode, In The Beginning, Samuel is being possessed by the YED and after Dean unwittingly tells him everything, he pins Dean to the wall and smells his neck while asking him if he’s one of the “special children.” According to Mitch Pileggi, the neck-smelling wasn’t rehearsed beforehand so Dean’s squicked-out reaction is completely genuine and the sexual subtext is even more creepy.

Another example is Misha Collin’s first appearance as Castiel. The staring and ignoring Dean’s personal space was not scripted, so that Jensen Ackles was genuinly surprised.

And in Bad Day at Black Rock, Dean’s final frustrated “SON OF A BITCH!” was an ad-lib by Jensen Ackles, and you can clearly see Jared Padalecki breaking character and laughing